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Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033
4To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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or the activity of eIF2B were noted
among treatment groups. Serum insulin was elevated 2.6- and 3.7-fold in
CHO and CL, respectively, but was not different in L, compared with S
(P < 0.05). These results suggest that leucine
stimulates protein synthesis in skeletal muscle by enhancing eIF4F
formation independently of increases in serum insulin.
KEY WORDS: • leucine • protein synthesis • translation initiation • skeletal muscle • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 1983
, Svanberg et al. 1996
, Yoshizawa et al. 1998
). The signals
responsible for stimulating muscle protein synthesis remain unclear;
however, several studies have suggested a role for amino acids in
mediating this anabolic response (Gautsch et al. 1998
,
Preedy and Garlick 1986
, Svanberg et al. 1997
, Yoshizawa et al. 1995
and 1998
). In
particular, a number of investigators reported that the
branched-chain amino acid, leucine, is unique in its ability to
independently stimulate muscle protein synthesis in vitro (Buse and Reid 1975
, Hong and Layman 1984
, Li and Jefferson 1978
). Further, in a recent study, we reported
that oral administration of leucine increases protein synthesis in
skeletal muscle of postabsorptive rats after exercise (Anthony et al. 1999
). The mechanism for stimulation of muscle protein
synthesis after leucine administration remains to be elucidated;
however, studies in vitro (Buse and Reid 1975
,
Buse et al. 1979
, Kimball et al. 1998b
)
and in perfused hindlimb preparations (Li and Jefferson 1978
) have suggested that leucine exerts its effects via
enhanced rates of mRNA translation.
The initiation of mRNA translation is a complex process requiring
several steps and more than a dozen eukaryotic initiation factors
(eIF)5
(reviewed by Pain 1996
, Voorma et al. 1994
). Two steps in the initiation pathway are subject to
regulation in vivo as follows: 1) the binding of initiator
methionyl-tRNA (met-tRNAi) to the 40 S
ribosomal subunit and 2) the binding of mRNA to the 43 S
preinitiation complex. In the first step,
met-tRNAi binds to the 40 S ribosomal subunit as
a ternary complex with eIF2 and GTP. Subsequently, the GTP bound to
eIF2 is hydrolyzed to GDP, and eIF2 is released from the ribosomal
subunit as a complex with GDP. For eIF2 to participate in another round
of initiation, it must exchange GDP for GTP before formation of a new
ternary complex can occur. A second initiation factor, eIF2B, mediates
guanine nucleotide exchange on eIF2. Inhibition of eIF2B activity
results in a decrease in the amount of eIF2·GTP available to form the
ternary complex, thereby restraining translation initiation. eIF2B
activity is regulated reciprocally in part by phosphorylation of eIF2.
Phosphorylation of the -subunit of eIF2 converts eIF2 from a
substrate to a competitive inhibitor of eIF2B (Kimball and Jefferson 1994
).
The binding of mRNA to the 43 S preinitiation complex requires a group
of proteins collectively referred to as eIF4F. eIF4F is a multisubunit
complex consisting of the following: 1) eIF4A, a RNA
helicase that functions in conjunction with another protein, eIF4B, to
unwind secondary structure in the 5'-untranslated region of the mRNA;
2) eIF4E, a protein that binds the
m7GTP cap present at the 5'-end of the mRNA; and
3) eIF4G, a large, 220-kDa polypeptide that functions as a
scaffold for eIF4E, eIF4A, the mRNA (via association with eIF4E) and
the ribosome (via association with eIF3). Collectively, the eIF4F
complex serves to recognize, unfold and guide the mRNA to the 43S
preinitiation complex (Pain 1996
).
Formation of an active eIF4F complex is influenced by alterations in
either the phosphorylation state or the availability of eIF4E.
Phosphorylation of eIF4E is suggested to stimulate translation rates
via increased association with eIF4G and eIF4A (Morley et al. 1993
) and/or increased mRNA cap-binding affinity
(Minich et al. 1994
). Alternatively, the availability of
eIF4E for eIF4F complex formation appears to be regulated by the
translational repressor, eIF4E-binding protein 1 (4E-BP1) (Pause et al. 1994
). 4E-BP1 competes with eIF4G for binding eIF4E and
is able to sequester eIF4E into an inactive complex. The binding of
4E-BP1 to eIF4E is regulated by phosphorylation of 4E-BP1, with
increased phosphorylation of the protein causing a decrease in the
affinity of 4E-BP1 for eIF4E.
The objective of this study was to investigate the mechanism by which an oral dose of leucine promotes protein synthesis in skeletal muscle of food-deprived rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The animal facilities and protocol were reviewed and approved by the Institutional Animal Care and Use Committee of the Pennsylvania State University, College of Medicine. Male Sprague-Dawley rats (~200 g) were maintained on a 12-h light:dark cycle with food (Harlan-Teklad Rodent Chow, Madison, WI) and water provided freely. The feed contained ~24% protein and 4% fat.
Rats were either freely fed (F) or food deprived for 18 h. Food-deprived rats were randomly assigned to one of four groups, i.e., not refed, but administered saline (S), or administered a 100% carbohydrate meal (CHO), a 100% leucine meal (L) or a combination of carbohydrate plus leucine (CL). The dose for all experimental meals was 5 mL provided by oral gavage. S rats were fed 5 mL saline (0.155 mol/L). Both freely fed and food-deprived rats were allowed free access to water, but no food was available to the food-deprived rats beyond the defined experimental meals.
The carbohydrate meal provided 2.63 g of carbohydrate and
consisted of 262.5 g/L glucose and 262.5 g/L sucrose in distilled
water. The leucine meal provided 0.27 g of leucine prepared as
54.0 g/L L-leucine in distilled water. The amount of
leucine given was equivalent to the amount of leucine consumed by rats
of this age and strain during 24 h of free access to an AIN-93
powdered diet (Harlan-Teklad, Madison, WI) (Gautsch et al. 1998
). The carbohydrate plus leucine meal (235.5 g/L glucose,
235.5 g/L sucrose and 54.0 g/L leucine in distilled water) was
isocaloric with the carbohydrate meal and isonitrogenous with the
leucine meal. The carbohydrate meal and the carbohydrate plus leucine
meal supplied ~15% of daily energy intake for this age and strain of
rat (Gautsch et al. 1998
).
A metabolic tracer consisting of a flooding dose (1.0 mL/100 g body
weight) of L-[2,3,4,5,6-3H] phenylalanine
(150 mmol/L containing 3.70 GBq/L) was injected via the tail vein 50
min after meal administration for the measurement of synthesis of total
mixed proteins in skeletal muscle (Garlick et al. 1980
).
Exactly 1 h after meal administration, rats were killed by
decapitation. Trunk blood was collected and centrifuged at 1800 x g for 10 min to obtain serum. The right gastrocnemius
and plantaris were excised as a unit for the measurement of skeletal
muscle protein synthesis and quickly frozen in liquid nitrogen. The
contralateral muscles were excised similarly and divided into two
parts. One portion was weighed, and homogenized in 7 vol of buffer
consisting of (mmol/L) 20
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (pH 7.4), 100 KCl, 0.2 EDTA, 2 ethylene glycol-bis(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 1
dithiothreitol, 50 sodium fluoride, 50 ß-glycerophosphate, 0.1
phenylmethylsulfonyl fluoride, 1 benzamidine and 0.5 sodium vanadate.
The homogenate was immediately centrifuged at 10,000 x g for 10 min at 4°C. The supernatant was used for
measurement of eIF distribution and phosphorylation as described below.
The other portion of hindlimb muscle was used to determine eIF2B
activity. All serum and tissue samples were stored at -80°C.
Serum measurements.
Serum insulin concentrations were analyzed using a commercial RIA kit
for rat insulin (Linco Research, St. Charles, MO). Serum leucine
concentrations were determined using reversed-phase HPLC after
precolumn derivatization of amino acids with
o-phthaldialdehyde as described previously (Furst et al. 1990
).
Measurement of skeletal muscle protein synthesis.
Fractional rates of skeletal muscle protein synthesis were estimated
from the rate of incorporation of radioactive phenylalanine into muscle
protein using the specific radioactivity of serum phenylalanine as
representative of the precursor pool (Kimball et al. 1992
). The elapsed time from injection of the metabolic tracer
until freezing of muscle in liquid nitrogen was recorded as the actual
time for incorporation of the radiolabeled amino acid into protein.
Quantitation of phosphorylated and unphosphorylated eIF2.
eIF2 was immunoprecipitated from aliquots of 10,000 x g supernatants using an anti-eIF2 monoclonal
antibody. The proportion of eIF2 present in the phosphorylated form
was determined by protein immunoblot analysis after separation of the
phosphorylated and unphosphorylated forms of the protein using slab gel
isoelectric focusing electrophoresis as described previously
(Kimball et al. 1998b
).
Quantitation of 4E-BP1·eIF4E and eIF4G·eIF4E complexes.
eIF4E was immunoprecipitated from 10,000 x g
supernatants using a monoclonal antibody to eIF4E (Kimball et al. 1997
). Next, samples were subjected to immunoblot analysis
using either a monoclonal antibody to 4E-BP1 or a polyclonal antibody
to eIF4G to determine the association of 4E-BP1 and eIF4G with eIF4E,
respectively, as described previously (Kimball et al. 1997
). Results were normalized to the amount of eIF4E in the
immunoprecipitate.
Quantitation of phosphorylated and unphosphorylated eIF4E.
The phosphorylated and unphosphorylated forms of eIF4E were separated
by isoelectric focusing of 10,000 x g supernatants
on a slab gel and quantitated by protein immunoblot analysis as
described previously (Kimball et al. 1997
).
Examination of 4E-BP1 phosphorylation state.
4E-BP1 was immunoprecipitated from 10,000 x g
supernatants of skeletal muscle with an anti-4E-BP1 monoclonal antibody
and subjected to protein immunoblot analysis as described previously
(Kimball et al. 1997
).
Phosphorylation of p70S6k.
Phosphorylation of the 70-kDa ribosomal protein S6 kinase,
p70S6k, was determined in 10,000 x g
supernatants by protein immunoblot analysis as previously described
(Gautsch et al. 1998
).
Measurement of eIF2B activity.
The guanine nucleotide exchange activity of eIF2B in skeletal muscle
was measured by the exchange of [3H]GDP bound to eIF2 for
nonradioactively labeled GDP as described previously (Kimball and Jefferson 1988
).
Statistical analysis.
All data were analyzed by the STATISTICA software package for the Macintosh, volume II (StatSoft, Tulsa, OK). Data were analyzed using a one-way ANOVA to assess main effects with treatment group (nutritional status + meal) as the independent variable. Serum leucine was analyzed using a one-way ANOVA after logarithmic transformation of the data. When a significant overall effect was detected, differences among individual means were assessed with Duncan’s Multiple Range post-hoc test. The level of significance was set at P < 0.05 for all statistical tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) to 23% of the values measured in freely fed rats (S vs. F). Oral
administration of carbohydrate (CHO) or a combination of carbohydrate
plus leucine (CL) resulted in serum insulin values that were 2.6- and
3.7-fold greater, respectively, than those of food-deprived rats
(S). In contrast, administration of leucine alone (L) did not increase
serum insulin concentrations relative to rats that were food deprived.
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). In contrast, administration of carbohydrate (CHO)
lowered serum leucine to 46% of the values measured in
food-deprived rats (S). Although the amounts of leucine in L and CL
were the same, administration of these meals did not result in
equivalent serum leucine concentrations. Serum leucine in rats provided
leucine alone was 16-fold greater than in S. In contrast, circulating
concentrations of leucine in rats administered the carbohydrate plus
leucine meal were stimulated 5.5-fold. Provision of carbohydrate may
have slowed intestinal absorption of the amino acid, resulting in
reduced serum leucine concentrations in CL compared with L.
Alternatively, lower serum leucine in CL may be attributed to a higher
rate of leucine disposal. Regardless of the basis for the difference in
serum leucine concentrations between L and CL, protein synthesis was
stimulated equally after administration of either
leucine-containing meal (see below).
Protein synthesis (Table 1)
was depressed to 65% of values observed in
skeletal muscle from rats that had been previously freely fed after
18 h of food deprivation (S vs. F). Provision of carbohydrate
alone did not affect muscle protein synthesis. In contrast,
administration of either leucine alone or the combination of
carbohydrate plus leucine stimulated complete recovery of muscle
protein synthesis. In both cases (L and CL), rates of protein synthesis
were not different from values in freely fed controls.
To examine potential mechanisms responsible for stimulating protein
synthesis by leucine, we first examined the guanine nucleotide exchange
activity of eIF2B after meal administration. eIF2B activity was not
altered by 18 h of food deprivation or by provision of nutrients
(Table 1)
. Similarly, there were no differences observed in the
phosphorylation state of eIF2 among the groups (Fig. 1
). Skeletal muscle eIF2 was almost entirely in the dephosphorylated
state irrespective of the treatment group. Therefore, eIF2B activity
was not altered by changes in the nutritional status of the rats and
cannot explain the stimulatory effect of leucine on muscle protein
synthesis.
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). Provision of the carbohydrate meal did not alter the association of
4E-BP1 with eIF4E. In contrast, administration of either carbohydrate
plus leucine or leucine alone reduced the amount of 4E-BP1 present in
the eIF4E immunoprecipitate, resulting in values that were not
different from those of freely fed rats.
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, ß and 
,
representing the differentially phosphorylated forms of the protein.
The most highly phosphorylated form, i.e., the -form, exhibits the
slowest electrophoretic mobility and does not bind eIF4E. Therefore, in
this study, phosphorylation of 4E-BP1 was expressed as the percentage
of the protein in the -form (Fig. 3
). The total amount of 4E-BP1 did not differ among the groups (data not
shown). Food deprivation reduced the amount of 4E-BP1 in the -form
to 20% of values in freely fed controls. Rats fed carbohydrate alone
did not differ in 4E-BP1 phosphorylation compared with
food-deprived rats. In contrast, the amount of 4E-BP1 in the
-form in rats fed leucine (L and CL) was at least threefold greater
than in food-deprived rats. These data provide evidence that oral
administration of leucine stimulates phosphorylation of 4E-BP1.
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). Administration of carbohydrate alone did not promote the association
of eIF4E with eIF4G. In contrast, eIF4G·eIF4E complex formation was
twofold greater in rats fed leucine alone compared with S, resulting in
values that were not different from those of freely fed controls.
Administration of carbohydrate plus leucine also enhanced the
association of eIF4E with eIF4G equivalent to an intermediate value
that did not differ from all other groups. Collectively, these results
suggest that oral administration of leucine increases eIF4E
availability for the formation of the active eIF4F complex.
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). In this study, 18 h of food
deprivation resulted in a 29% increase in the amount of eIF4E found in
the phosphorylated form (Fig. 5
). Administration of carbohydrate alone did not alter the percentage of
eIF4E found in the phosphorylated form compared with food-deprived
rats. In contrast, provision of carbohydrate plus leucine or leucine
alone resulted in a partial dephosphorylation of eIF4E, yielding values
that were not different from those in freely fed rats. Therefore, our
data indicate that oral administration of leucine promotes the partial
dephosphorylation of eIF4E in skeletal muscle.
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) or leucine (Kimball et al. 1998b
) occur
via one or more pathways that also result in the activation of the
70-kDa ribosomal protein S6 kinase, p70S6k.
p70S6k has been implicated in stimulating protein
synthesis under conditions that promote 4E-BP1 phosphorylation
(Sonenberg 1996
). Phosphorylation of the
p70S6k is associated with its activation
(Cheatham et al. 1994
, Chung et al. 1994
). After SDS-PAGE, p70S6k
resolves into multiple electrophoretic forms, with increased
phosphorylation corresponding to decreased electrophoretic mobility.
Only the most highly phosphorylated forms of the kinase are associated
with increased p70S6k activity in vivo
(Weng et al. 1998
). Food deprivation resulted in greater
electrophoretic mobility, suggesting that p70S6k
activation was reduced compared with freely fed controls (Fig. 6
). Provision of carbohydrate alone promoted a slight shift to a more
slowly migrating form. Administration of leucine or carbohydrate plus
leucine further enhanced phosphorylation of the kinase and resulted in
bands with the slowest electrophoretic mobility. These results suggest
that leucine activates p70S6k activity in muscle
of food-deprived rats independent of increases in serum insulin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a recent study, we reported that oral administration of leucine,
alone or in combination with carbohydrate, immediately after treadmill
exercise restores rates of protein synthesis to values equivalent to
those of freely fed controls 1 h after meal provision
(Anthony et al. 1999
). Similarly, in this study, leucine
stimulated muscle protein synthesis in rats that had been food deprived
for 18 h. In both studies, serum insulin concentrations in rats
fed leucine alone was not different from that of food-deprived
controls. Collectively, these results suggest that the anabolic effect
of leucine does not require an elevation in circulating insulin
concentrations.
These results do not imply, however, that the protein synthetic
response to leucine is independent of circulating insulin
concentrations. Indeed, intraperitoneal administration of antibodies to
insulin 3 h before and immediately after the start of refeeding
into mice deprived of food overnight caused a partial attenuation in
the stimulation of muscle protein synthesis (Svanberg et al. 1996
). Furthermore, Preedy and Garlick (1986)
reported that intravenous administration of anti-insulin serum to
food-deprived rats immediately before refeeding prevented a
postprandial rise in muscle protein synthesis. Therefore, insulin
availability may be essential to facilitate increases in protein
synthesis after amino acid intake.
There are several potential mechanisms that may lead to leucine’s
stimulation of protein synthesis. Both the number of ribosomes per cell
and the translational efficiency per ribosome determine the rate of
tissue protein synthesis. In growing rats, comparable in age and strain
to those employed in this study, food deprivation for up to 3 d
and subsequent refeeding did not alter RNA content in skeletal muscle
(Nakano and Sidransky 1978
). Because >80% of total
muscle RNA is ribosomal, these results suggest that short-term
alterations in nutritional status of the animal do not alter ribosome
number. Rather, alterations in the rate of protein synthesis in
skeletal muscle that occur with acute modulation of nutritional status
parallel changes in translation initiation activity (Yoshizawa et al. 1995
).
Formation of the ternary initiation complex is one rate-controlling
step in the complex process of translation initiation. This step
requires interaction between eIF2 and eIF2B. Few studies have examined
the role of eIF2/eIF2B in modulating skeletal muscle protein synthesis
after food deprivation or refeeding. Cox et al. (1988)
demonstrated that 48 h of starvation is without effect on eIF2B
activity or on phosphorylation of eIF2 even though muscle protein
synthesis is decreased. Additionally, Yoshizawa et al. (1997)
observed no change in activity of eIF2B or the
proportion of eIF2 in the phosphorylated form when refeeding a
macronutrient-mixed meal to rats that had been food deprived for18
h. Similarly, in this study, neither food deprivation nor meal
administration altered the activity of eIF2B or the proportion of
eIF2 in the phosphorylated form. These results suggest that the
stimulatory effects of refeeding leucine on skeletal muscle protein
synthesis in food-deprived rats must be through modulation of
translational components other than those that are requisite for
formation of the ternary initiation complex.
Another principal site of regulation in the initiation process involves
the recognition and unwinding of the mRNA to allow binding to the 40S
ribosome. This tightly regulated step requires the participation of
eIF4B and eIF4F, a three-subunit complex consisting of eIF4A, eIF4E
and eIF4G. There are currently two characterized mechanisms in which
changes in eIF4F function alter translation initiation. The first
mechanism involves modulation of eIF4E availability via the binding of
eIF4E to the translational repressor, 4E-BP1. Studies in rats show that
an increased proportion of eIF4E is bound to 4E-BP1 rather than eIF4G
after overnight food deprivation (Yoshizawa et al. 1997
). The changes in eIF4E availability occur in response to a
decrease in 4E-BP1 phosphorylation. These results suggest that food
deprivation decreases the availability of eIF4E to form an active
eIF4G·eIF4E complex, and hence translation initiation. Refeeding
starved animals a nutritionally complete diet elicits the opposite
effect, i.e., hyperphosphorylation of 4E-BP1, freeing eIF4E from 4E-BP1
and increasing formation of eIF4G·eIF4E (Yoshizawa et al. 1997
and 1998
). Therefore, refeeding is postulated to augment rates
of protein synthesis by promoting increases in eIF4E availability. The
results of this study demonstrate that the stimulatory effect of
leucine on muscle protein synthesis is also associated with an increase
in eIF4E availability. Oral administration of meals containing leucine
stimulated the phosphorylation of 4E-BP1, reduced its association with
eIF4E and resulted in a greater proportion of eIF4E free to interact
with eIF4G. These results implicate leucine as a potential signaling
molecule that promotes eIF4E availability after refeeding.
A second mechanism of altered eIF4F function involves modulation of
eIF4E phosphorylation. Studies using cells in culture suggest that an
increase in eIF4E phosphorylation enhances mRNA cap-binding
affinity and/or association with eIF4G. These changes augment rates of
protein synthesis and cell growth (Bu et al. 1993
,
Minich et al. 1994
). In contrast, experiments in vivo
demonstrate that eIF4E phosphorylation either does not change or
increases and then decreases during food deprivation and after
refeeding, respectively (Yoshizawa et al. 1997
and 1998
). In this study, administration of test diets containing
leucine, but not carbohydrate alone, resulted in a net
dephosphorylation of eIF4E compared with food-deprived rats. The
basis for this reduction in phosphorylation is unknown. It has been
proposed that the rate of phosphate turnover on eIF4E rather than the
actual amount of eIF4E in the phosphorylated form is important for
controlling protein synthesis (Rinker-Schaeffer et al. 1992
). Supporting this notion, in insulin-treated cells in
culture, the magnitude of the increase of
32Pi incorporation into
eIF4E caused by insulin is much greater than the increase in the
proportion of the protein in the phosphorylated form
(Rinker-Schaeffer et al. 1992
, Rychlik et al. 1990
).
The precise intracellular signaling pathway responsible for increasing
rates of translation initiation in response to oral administration of
leucine remains to be elucidated, but recent evidence suggests the
involvement of p70S6k (Kimball et al.
1998, Patti et al. 1998
). The
p70S6k signaling pathway is implicated in stimulating
skeletal muscle protein synthesis and increasing 4E-BP1 phosphorylation
in response to refeeding (Svanberg et al. 1997
).
Further, in cultures of L6 myoblasts, addition of leucine to the medium
of cells deprived of the amino acid increases protein synthesis and
4E-BP1 phosphorylation (Kimball et al. 1998b
).
Concomitant with these changes, leucine also increases phosphorylation
of p70S6k. This study supports these observations because
oral administration of leucine to food-deprived rats increased the
degree of p70S6k phosphorylation, suggesting that the amino
acid augments rates of protein synthesis through the activation of the
p70S6k signaling pathway.
Although the p70S6k signaling pathway appears to be
important in regulating rates of translation initiation,
p70S6k does not appear to be the kinase responsible for
phosphorylating 4E-BP1 directly because purified p70S6k
does not phosphorylate recombinant 4E-BP1 directly (Haystead et al. 1994
). In contrast, the kinase, mammalian target of
rapamycin (mTOR), phosphorylates both 4E-BP1 and p70S6k in
vitro (Burnett et al. 1998
). Studies using L6 myoblasts
in culture treated with the immunosuppressant drug, rapamycin, an
inhibitor of mTOR, show that it blocks activation of p70S6k
as well as phosphorylation of 4E-BP1 caused by leucine (Kimball et al. 1999
). Whether oral administration of leucine activates
mTOR directly or promotes phosphorylation of 4E-BP1 and
p70S6k via another kinase(s) remains to be determined.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by research grants DK-15658 (L.S.J.), GM-39277 (T.C.V.) and a training grant, GM-08619, (supports J.C.A.) from the National Institutes of Health.
3 J.C.A. received the 1999 ASNS/Procter and Gamble Company Graduate Student Research Award.
5 Abbreviations used: CHO, food-deprived rats refed a 100% carbohydrate meal; CL, food-deprived rats refed a combination of carbohydrate plus leucine; 4E-BP1, eIF4E-binding protein 1; eIF, eukaryotic initiation factor; F, freely fed rats; L, food-deprived rats refed a 100% leucine meal; met-tRNAi, initiator methionyl-tRNA; mTOR, mammalian target of rapamycin kinase; p70S6k, 70-kDa ribosomal protein S6 kinase; S, food-deprived rats.
Manuscript received June 8, 1999. Initial review completed July 14, 1999. Revision accepted October 19, 1999.
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J. C. Anthony, C. H. Lang, S. J. Crozier, T. G. Anthony, D. A. MacLean, S. R. Kimball, and L. S. Jefferson Contribution of insulin to the translational control of protein synthesis in skeletal muscle by leucine Am J Physiol Endocrinol Metab, May 1, 2002; 282(5): E1092 - E1101. [Abstract] [Full Text] [PDF]
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S. R. Kimball Regulation of Global and Specific mRNA Translation by Amino Acids J. Nutr., May 1, 2002; 132(5): 883 - 886. [Abstract] [Full Text] [PDF]
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 J. C. Anthony, A. K. Reiter, T. G. Anthony, S. J. Crozier, C. H. Lang, D. A. MacLean, S. R. Kimball, and L. S. Jefferson Orally Administered Leucine Enhances Protein Synthesis in Skeletal Muscle of Diabetic Rats in the Absence of Increases in 4E-BP1 or S6K1 Phosphorylation Diabetes, April 1, 2002; 51(4): 928 - 936. [Abstract] [Full Text] [PDF]
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Y. Boirie, K. R. Short, B. Ahlman, M. Charlton, and K. S. Nair Tissue-Specific Regulation of Mitochondrial and Cytoplasmic Protein Synthesis Rates by Insulin Diabetes, December 1, 2001; 50(12): 2652 - 2658. [Abstract] [Full Text] [PDF]
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T. C. Vary, L. S. Jefferson, and S. R. Kimball Insulin fails to stimulate muscle protein synthesis in sepsis despite unimpaired signaling to 4E-BP1 and S6K1 Am J Physiol Endocrinol Metab, November 1, 2001; 281(5): E1045 - E1053. [Abstract] [Full Text] [PDF]
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T. G. Anthony, A. K. Reiter, J. C. Anthony, S. R. Kimball, and L. S. Jefferson Deficiency of dietary EAA preferentially inhibits mRNA translation of ribosomal proteins in liver of meal-fed rats Am J Physiol Endocrinol Metab, September 1, 2001; 281(3): E430 - E439. [Abstract] [Full Text] [PDF]
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J. S. Greiwe, G. Kwon, M. L. McDaniel, and C. F. Semenkovich Leucine and insulin activate p70 S6 kinase through different pathways in human skeletal muscle Am J Physiol Endocrinol Metab, September 1, 2001; 281(3): E466 - E471. [Abstract] [Full Text] [PDF]
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M. Balage, S. Sinaud, M. Prod'Homme, D. Dardevet, T. C. Vary, S. R. Kimball, L. S. Jefferson, and J. Grizard Amino acids and insulin are both required to regulate assembly of the eIF4E {middle dot} eIF4G complex in rat skeletal muscle Am J Physiol Endocrinol Metab, September 1, 2001; 281(3): E565 - E574. [Abstract] [Full Text] [PDF]
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L. S. Jefferson and S. R. Kimball Amino Acid Regulation of Gene Expression J. Nutr., September 1, 2001; 131(9): 2460S - 2466. [Abstract] [Full Text] [PDF]
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E. Blomstrand and B. Saltin BCAA intake affects protein metabolism in muscle after but not during exercise in humans Am J Physiol Endocrinol Metab, August 1, 2001; 281(2): E365 - E374. [Abstract] [Full Text] [PDF]
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Z. Liu, L. A. Jahn, W. Long, D. A. Fryburg, L. Wei, and E. J. Barrett Branched Chain Amino Acids Activate Messenger Ribonucleic Acid Translation Regulatory Proteins in Human Skeletal Muscle, and Glucocorticoids Blunt This Action J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2136 - 2143. [Abstract] [Full Text]
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T. G. Anthony, J. C. Anthony, F. Yoshizawa, S. R. Kimball, and L. S. Jefferson Oral Administration of Leucine Stimulates Ribosomal Protein mRNA Translation but Not Global Rates of Protein Synthesis in the Liver of Rats J. Nutr., April 1, 2001; 131(4): 1171 - 1176. [Abstract] [Full Text]
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 G. GARIBOTTO, A. SOFIA, A. CANEPA, S. SAFFIOTI, P. SACCO, M. R. SALA, L. DERTENOIS, N. PASTORINO, G. DEFERRARI, and R. RUSSO Acute Effects of Peritoneal Dialysis with Dialysates Containing Dextrose or Dextrose and Amino Acids on Muscle Protein Turnover in Patients with Chronic Renal Failure J. Am. Soc. Nephrol., March 1, 2001; 12(3): 557 - 567. [Abstract] [Full Text]
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S. M. Hutson and R. A. Harris Introduction J. Nutr., March 1, 2001; 131(3): 839S - 840. [Full Text]
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R. A. Harris, R. Kobayashi, T. Murakami, and Y. Shimomura Regulation of Branched-Chain {{alpha}}-Keto Acid Dehydrogenase Kinase Expression in Rat Liver J. Nutr., March 1, 2001; 131(3): 841S - 845. [Abstract] [Full Text]
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J. C. Anthony, T. G. Anthony, S. R. Kimball, and L. S. Jefferson Signaling Pathways Involved in Translational Control of Protein Synthesis in Skeletal Muscle by Leucine J. Nutr., March 1, 2001; 131(3): 856S - 860. [Abstract] [Full Text]
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C. J. Lynch Role of Leucine in the Regulation of mTOR by Amino Acids: Revelations from Structure-Activity Studies J. Nutr., March 1, 2001; 131(3): 861S - 865. [Abstract] [Full Text]
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E. Volpi, B. Mittendorfer, B. B. Rasmussen, and R. R. Wolfe The Response of Muscle Protein Anabolism to Combined Hyperaminoacidemia and Glucose-Induced Hyperinsulinemia Is Impaired in the Elderly J. Clin. Endocrinol. Metab., December 1, 2000; 85(12): 4481 - 4490. [Abstract] [Full Text]
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C. H. Lang, R. A. Frost, V. Kumar, and T. C. Vary Impaired myocardial protein synthesis induced by acute alcohol intoxication is associated with changes in eIF4F Am J Physiol Endocrinol Metab, November 1, 2000; 279(5): E1029 - E1038. [Abstract] [Full Text] [PDF]
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S. R. Kimball, L. S. Jefferson, H. V. Nguyen, A. Suryawan, J. A. Bush, and T. A. Davis Feeding stimulates protein synthesis in muscle and liver of neonatal pigs through an mTOR-dependent process Am J Physiol Endocrinol Metab, November 1, 2000; 279(5): E1080 - E1087. [Abstract] [Full Text] [PDF]
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O. J. Shah, J. C. Anthony, S. R. Kimball, and L. S. Jefferson Glucocorticoids oppose translational control by leucine in skeletal muscle Am J Physiol Endocrinol Metab, November 1, 2000; 279(5): E1185 - E1190. [Abstract] [Full Text] [PDF]
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D. Dardevet, C. Sornet, M. Balage, and J. Grizard Stimulation of In Vitro Rat Muscle Protein Synthesis by Leucine Decreases with Age J. Nutr., November 1, 2000; 130(11): 2630 - 2635. [Abstract] [Full Text] [PDF]
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O. J. Shah, J. C. Anthony, S. R. Kimball, and L. S. Jefferson 4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle Am J Physiol Endocrinol Metab, October 1, 2000; 279(4): E715 - E729. [Abstract] [Full Text] [PDF]
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J. C. Anthony, F. Yoshizawa, T. G. Anthony, T. C. Vary, L. S. Jefferson, and S. R. Kimball Leucine Stimulates Translation Initiation in Skeletal Muscle of Postabsorptive Rats via a Rapamycin-Sensitive Pathway J. Nutr., October 1, 2000; 130(10): 2413 - 2419. [Abstract] [Full Text]
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L. J. C. van Loon, M. Kruijshoop, H. Verhagen, W. H. M. Saris, and A. J. M. Wagenmakers Ingestion of Protein Hydrolysate and Amino Acid-Carbohydrate Mixtures Increases Postexercise Plasma Insulin Responses in Men J. Nutr., October 1, 2000; 130(10): 2508 - 2513. [Abstract] [Full Text]
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S. R. Kimball, P. A. Farrell, H. V. Nguyen, L. S. Jefferson, and T. A. Davis Developmental decline in components of signal transduction pathways regulating protein synthesis in pig muscle Am J Physiol Endocrinol Metab, March 1, 2002; 282(3): E585 - E592. [Abstract] [Full Text] [PDF]
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